U.S. patent application number 12/306446 was filed with the patent office on 2009-10-08 for use of stilbene derivatives for treatment and prevention of aquatic mold infections.
Invention is credited to Rama Falk, Itzhack Polacheck, Simon Tinman.
Application Number | 20090252768 12/306446 |
Document ID | / |
Family ID | 38846097 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090252768 |
Kind Code |
A1 |
Polacheck; Itzhack ; et
al. |
October 8, 2009 |
USE OF STILBENE DERIVATIVES FOR TREATMENT AND PREVENTION OF AQUATIC
MOLD INFECTIONS
Abstract
The invention relates to methods of treatment and prevention of
aquatic mold infections in aquatic organisms and methods of
disinfecting equipment used in raising aquatic organisms. The
methods comprise use of one or more stilbene derivatives, including
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives.
Inventors: |
Polacheck; Itzhack;
(Jerusalem, IL) ; Tinman; Simon; (Bat Hefer,
IL) ; Falk; Rama; (Merchaviah, IL) |
Correspondence
Address: |
FENNEMORE CRAIG
3003 NORTH CENTRAL AVENUE, SUITE 2600
PHOENIX
AZ
85012
US
|
Family ID: |
38846097 |
Appl. No.: |
12/306446 |
Filed: |
June 27, 2007 |
PCT Filed: |
June 27, 2007 |
PCT NO: |
PCT/IL2007/000784 |
371 Date: |
May 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60816619 |
Jun 27, 2006 |
|
|
|
Current U.S.
Class: |
424/400 ; 422/28;
514/236.2; 514/245 |
Current CPC
Class: |
A61K 31/53 20130101;
A61P 31/00 20180101; Y02A 50/30 20180101; A61P 33/00 20180101; Y02A
50/492 20180101 |
Class at
Publication: |
424/400 ;
514/245; 422/28; 514/236.2 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 31/53 20060101 A61K031/53; A61L 2/18 20060101
A61L002/18; A61K 31/5377 20060101 A61K031/5377 |
Claims
1. A method of preventing or treating an oomycete infection in an
aquatic organism, the method comprising the step of contacting the
aquatic organism with an effective amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative of Formula (I): ##STR00003## wherein R.sub.1 and R.sub.2
are the same or different and are each independently selected from
the group consisting of SO.sub.3H, SO.sub.3Na, SO.sub.3K,
SO.sub.3NH.sub.4 and H; R.sub.3 to R.sub.10 are the same or
different and are selected from the group consisting of H; linear
or branched C.sub.1-C.sub.6 alkyl; linear or branched
C.sub.2-C.sub.6 alkenyl, wherein said alkyl or alkenyl are each
independently unsubstituted or substituted with a hydroxyl,
carboxyl, or carboxamide group; phenyl; and phenyl substituted with
R.sub.1 or R.sub.2 wherein R.sub.1 and R.sub.2 are as defined
above; or one or more of R.sub.3 and R.sub.4, R.sub.5 and R.sub.6,
R.sub.7 and R.sub.8 or R.sub.9 and R.sub.10, together with the
nitrogen to which they are attached, form a heterocyclic ring which
can further comprise one or more additional heteroatoms selected
from N, O and S; and salts, hydrates, solvates and polymorphs
thereof; thereby preventing or treating the oomycete infection in
said aquatic organism.
2. The method of claim 1, wherein two or more of R.sub.3, R.sub.4,
R.sub.7 and R.sub.8 are the same and are selected from the group
consisting of CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3,
CH.sub.2CH.sub.2CONH.sub.2, CH.sub.3 and H; or one or more of
R.sub.3 and R.sub.4, R.sub.7 and R.sub.8 together with the nitrogen
to which they are attached, form a morpholinyl ring.
3. The method of claim 1, wherein two or more of R.sub.5, R.sub.6,
R.sub.9 and R.sub.10 are the same and are selected from the group
consisting of phenyl and phenyl substituted with SO.sub.3Na.
4. The method of claim 1, wherein R.sub.1 and R.sub.2 are the same
and are selected from the group consisting of SO.sub.3H and
SO.sub.3Na, or wherein R.sub.1 and R.sub.2 are different and are
selected from the group consisting of SO.sub.3H, SO.sub.3Na,
SO.sub.3K, SO.sub.3NH.sub.4 and H.
5. (canceled)
6. The method of claim 1, wherein the
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is selected from the group consisting of
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonic acid; disodium
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonate; potassium sodium
4,4'-bis-(6-anilino-4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino-
)stilbene-2,2'-disulfonate; 2,2'-stilbenedisulfonic acid,
4,4'-bis-(4-anilino-6-((2-hydroxyethyl)methylamino)-s-triazin-2-yl)amino)-
-, disodium salt; disodium
4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-d-
isulfonate; tetrasodium
4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulfonatoanilino)-1,3,5-triaz-
in-2-yl]amino]stilbene-2,2'-disulfonate; tetrasodium
4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,-
5-triazin-2-yl]amino]-stilbene-2,2'-disulfonate; and
2,2'-stilbenedisulfonic acid,
4,4'-bis-[[4-[(2-carbamoylethyl)(2-hydroxylethyl)amino]-6-(p-sulfoanilino-
)-s-triazin-2-yl]amino]-, tetrasodium salt.
7. The method of claim 1, wherein the oomycete is selected from the
group consisting of Saprolegnia spp., Aphanomyces spp. and
Branchiomyces spp.
8. The method of claim 7, wherein the oomycete is Saprolegnia
parasitica.
9. The method of claim 1, wherein the aquatic organism is selected
from the group consisting of fish, fish eggs and shellfish.
10. The method of claim 9 wherein the fish is selected from the
group consisting of barramundi, bass, bream, carp, catfish, chub,
eel, elver, flounder, gilthead, guppy, halibut, koi, labrax,
mullet, paddlefish, plaice, pompano, redfish, red-drum, salmon,
sole, sturgeon, tilapia, trout, tuna and whitefish.
11. The method of claim 1, wherein the at least one
4,4'-bis(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is provided in a form selected from the group consisting
of a solution, a dissolving tablet, a gel and an impregnated
material.
12. The method of claim 11, wherein the wherein the at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is provided in the form of a solution at a concentration
of about 20 to about 200 mg/L.
13. The method of claim 12, wherein the at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives is present in the solution at a concentration of about
25 mg/L.
14. The method of claim 1, wherein the contacting step is for a
period of about 2 to about 16 hours, or for a period of about 8
hours.
15. (canceled)
16. The method of claim 14, wherein the contacting step is repeated
at 48 hour intervals.
17. The method of claim 1, wherein the oomycete infection is
concurrent with or accompanied by a parasitic infection, wherein
the parasitic infection is caused by at least one parasite selected
from the group consisting of Amyloodinium spp., Argulus spp.,
Ascocotyle spp., Bothricephalus spp., Camallanus spp., Capilaria
spp., Centrocestus spp., Chilodonella spp., Coccidia spp.,
Contracaecum spp., Cryptobia spp., Cryptocaryon spp., Dactylogyrus
spp., Dermocystidium spp., Ergasilus spp., Euclinostomum spp.,
Gyrodactylus spp., Hexamita spp., Ichtyobodo spp., Ichtyophtirius
spp., Lernaea spp., Metacercarius spp., Microsporidia spp.,
Myxosporea spp., Oodinium spp., Sanguinicola spp., Sessiline spp.,
Spironucleus spp., Tetrahymena spp., Trichodina spp., Trichodinella
spp. and Tripartiella spp.
18. (canceled)
19. A method of disinfecting equipment used for raising aquatic
organisms, the method comprising the step of contacting the
equipment with an effective amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative of Formula (I): ##STR00004## wherein R.sub.1 and R.sub.2
are the same or different and are each independently selected from
the group consisting of SO.sub.3H, SO.sub.3Na, SO.sub.3K,
SO.sub.3NH.sub.4 and H; and wherein R.sub.3 to R.sub.10 are the
same or different and are selected from the group consisting of H;
linear or branched C.sub.1-C.sub.6 alkyl; linear or branched
C.sub.2-C.sub.6 alkenyl, wherein said alkyl or alkenyl are each
independently unsubstituted or substituted with a hydroxyl,
carboxyl, or carboxamide group; phenyl; and phenyl substituted with
R.sub.1 or R.sub.2 wherein R.sub.1 and R.sub.2 are as defined
above; or one or more of R.sub.3 and R.sub.4, R.sub.5 and R.sub.6,
R.sub.7 and R.sub.8 or R.sub.9 and R.sub.10, together with the
nitrogen to which they are attached, form a heterocyclic ring which
can further comprise one or more additional heteroatoms selected
from N, O and S; and salts, hydrates, solvates and polymorphs
thereof; thereby disinfecting said equipment.
20. The method of claim 19, wherein two or more of R.sub.3,
R.sub.4, R.sub.7 and R.sub.8 are the same and are selected from the
group consisting of CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3,
CH.sub.2CH.sub.2CONH.sub.2, CH.sub.3 and H; or one or more of
R.sub.3 and R.sub.4, R.sub.7 and R.sub.8 together with the nitrogen
to which they are attached, form a morpholinyl ring.
21. The method of claim 19, wherein two or more of R.sub.5,
R.sub.6, R.sub.9 and R.sub.10 are the same and are selected from
the group consisting of phenyl and phenyl substituted with
SO.sub.3Na.
22. The method of claim 19, wherein R.sub.1 and R.sub.2 are the
same and are selected from the group consisting of SO.sub.3H and
SO.sub.3Na, or wherein R.sub.1 and R.sub.2 are different and are
selected from the group consisting of SO.sub.3H, SO.sub.3Na,
SO.sub.3K, SO.sub.3NH.sub.4 and H.
23. (canceled)
24. The method of claim 19, wherein the
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is selected from the group consisting of
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonic acid; disodium
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonate; potassium sodium
4,4'-bis-(6-anilino-4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino-
)stilbene-2,2'-disulfonate; 2,2'-stilbenedisulfonic acid,
4,4'-bis-(4-anilino-6-((2-hydroxyethyl)methylamino)-s-triazin-2-yl)amino)-
-, disodium salt; disodium
4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-d-
isulfonate; tetrasodium
4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulfonatoanilino)-1,3,5-triaz-
in-2-yl]amino]stilbene-2,2'-disulfonate; tetrasodium
4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,-
5-triazin-2-yl]amino]-stilbene-2,2'-disulfonate; and
2,2'-stilbenedisulfonic acid,
4,4'-bis-[[4-[(2-carbamoylethyl)(2-hydroxylethyl)amino]-6-(p-sulfoanilino-
)-s-triazin-2-yl]amino]-, tetrasodium salt.
25. The method of claim 19, wherein the equipment is contaminated
with at least one oomycete.
26. (canceled)
27. (canceled)
28. (canceled)
29. The method of claim 19, wherein the at least one
4,4'-bis(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is provided in a form selected from the group consisting
of a solution, a dissolving tablet, a gel and an impregnated
material.
30. The method of claim 29, wherein the at least one
4,4'-bis(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is provided in the form of a solution at a concentration
of about 20 to about 200 mg/L.
31. The method of claim 30, wherein the at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives is present in the solution at a concentration of about
25 mg/L.
32. The method of claim 19, wherein the contacting step is for a
period of about 2 to about 16 hours, or for a period of about 8
hours.
33. (canceled)
34. The method of claim 32, wherein the contacting is repeated at
48 hour intervals.
35. The method of claim 19, wherein the equipment is further
contaminated with at least one parasite selected from the group
consisting of Amyloodinium spp., Argulus spp., Ascocotyle spp.,
Bothricephalus spp., Camallanus spp., Capilaria spp., Centrocestus
spp., Chilodonella spp., Coccidia spp., Contracaecum spp.,
Cryptobia spp., Cryptocaryon spp., Dactylogyrus spp.,
Dermocystidium spp., Ergasilus spp., Euclinostomum spp.,
Gyrodactylus spp., Hexamita spp., Ichtyobodo spp., Ichtyophtirius
spp., Lernaea spp., Metacercarius spp., Microsporidia spp.,
Myxosporea spp., Oodinium spp., Sanguinicola spp., Sessiline spp.,
Spironucleus spp., Tetrahymena spp., Trichodina spp., Trichodinella
spp. and Tripartiella spp.
36. (canceled)
37. The method of claim 19, wherein the at least one oomycete is
selected from the group consisting of Saprolegnia spp., Aphanomyces
spp. and Branchiomyces spp.
Description
FIELD OF THE INVENTION
[0001] The invention relates to compositions of stilbene
derivatives useful in methods of treatment and prevention of mold
infections in aquatic organisms such as fish and fish eggs.
BACKGROUND OF THE INVENTION
[0002] Oomycetes (water molds) such as Saprolegnia, Branchiomyces
and Aphanomyces are responsible for devastating infections of fish
in aquaculture, fish farms and hobby fish tanks.
[0003] Members of the genus Saprolegnia cause saprolegniosis, a
disease that is characterized by visible white or grey patches of
filamentous mycelium on the body or fins of freshwater fish. If
untreated, infection with Saprolegnia species (spp.) leads to death
by haemodilution. Saprolegnia spp. also infect fish eggs by
adhesion to and penetration of the egg membrane. Saprolegnia spp.
are considered opportunistic pathogens that are saprophytes.
Infection frequently occurs during the winter, often resulting in
large-scale "winter kill" epidemics. Conditions which promote
proliferation of Saprolegnia spp. and their infectious zoospores
include abrupt decreases in water temperatures and high density
fish farming activities, and also appear to render fish vulnerable
to infection due to increased physiological stress and immune
system suppression.
[0004] Saprolegnia parasitica is one of the most economically
damaging fish pathogens, causing losses of millions of dollars
annually worldwide, particularly in the salmon and trout markets.
In addition to being an opportunistic pathogen, some S. parasitica
strains are highly virulent and cause primary infections.
[0005] Branchiomycosis is another infection occurring in fresh
water fish, primarily in carp and eel. It is caused by
Branchiomyces sanguinis and B. demigrans, with affected fish
showing prominent gill necrosis and respiratory distress. The
disease occurs most commonly in ponds with abundant organic matter,
and high ammonia levels.
[0006] Parasitic infections are often concurrent with or accompany
oomycete infections in commercial fish farming settings. Such
parasitic infections include those caused by Ichthyophthirius
multifilis, Trichodina spp., Dactylogyrus spp. and Gyrodactylus
spp.
[0007] Oomycetes, including Saprolegnia spp. and Branchiomyces spp.
are filamentous eukaryotic microorganisms which have many
fungus-like characteristics, but are not true fungi. Like the true
fungi, they feed on decaying matter and grow as branching filaments
with non-septate hyphae. However, their cell wall is not composed
of chitin (as in the true fungi) but is composed of a mixture of
cellulosic compounds and other .beta.-glucans. Further, oomycetes
have several clearly defined developmental stages that are not
found in fungi. Recent molecular studies have shown however, that
closely related virulence components are shared between oomycetes
and fungi.
[0008] Prior art methods for treatment of pathogenic oomycete
infections, including saprolegniosis and branchiomycosis suffer
from various disadvantages. The chemicals Diquat (a herbicide),
benzalkonium chlorides, copper sulfate and potassium permanganate
have all been disclosed to be useful for treatment of
branchiomycosis. None of these however, are approved by the U.S.
Food and Drug Administration for disease control in food
fishes.
[0009] U.S. Pat. No. 6,160,023 discloses use of bronopol
(2-bromo-nitropropane-1,3-diol) for treatment and prophylaxis of S.
parasitica infections in fish, and for disinfecting equipment used
in raising fish. This compound is mainly effective against
infection present in fish eggs, but not that occurring in fish. In
addition, it is relatively toxic to commercially important fish
species.
[0010] Malachite green
(4-[(4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline)
was previously widely used to control saprolegniosis. While this
organic dye is very efficient at killing S. parasitica, its use has
been banned since 2002 around the world, due to its carcinogenic,
teratogenic and toxicological properties. This has resulted in a
dramatic increase of Saprolegnia infections in commercial settings.
Therefore, there is an urgent need for novel alternative methods of
management of saprolegniosis.
[0011] Methods are also known for controlling parasitic infections
in fish. U.S. Pat. No. 5,464,837 and U.S. Pat. No. 5,188,832
disclose use of triazine dione compounds; U.S. Pat. No. 5,313,911
discloses use of hydrogen peroxide; U.S. Pat. No. 6,054,454
discloses use of oxadiazine derivatives; U.S. Pat. No. 6,982,285
discloses use of benzoylurea derivatives; U.S. Pat. No. 6,117,457
discloses use of peracetic acid; U.S. Pat. No. 5,593,678 discloses
use of orthovanadate salts; U.S. Pat. No. 5,504,081 discloses use
of nitromethylene derivatives. None of these methods are known to
be useful for simultaneous control of oomycete and parasitic
infections.
[0012] Stilbene derivatives, including
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives, are known fluorescent brightening agents which bind to
polysaccharides having .beta.-glucosidic linkages, including
cellulose and chitin. Such compounds show antifungal activity,
presumably due to interaction with and disruption of chitin
microfibrils which constitute a major structural element in the
fungal cell wall (Roncero et al. (1985) J. Bacteriol.
163:1180-1185). Antifungal effects of fluorescent brighteners have
been demonstrated in true fungi which contain chitin in their cell
walls, including plant pathogenic fungi (Seppanen et al. (2004)
Plant Cell. Rep. 22:584-593), human pathogenic fungi-like yeasts
(Roncero et al. (1988) J. Bacteriol. 170:1950-54), dermatophytes
(Inamori et al. (1985) Chem. Pharm. Bull. (Tokyo) 33:2904-9) and
red alga (Belliveau et al. (1990) Stain Technol. 65:303-311), but
not in aquatic molds which contain cellulose instead of chitin in
their cell walls.
[0013] U.S. Pat. No. 4,723,034 discloses 2-vinyl stilbene
derivatives useful as fungicides for plant protection and wood
preservation.
[0014] U.S. Pat. No. 5,359,131 and U.S. Pat. No. 5,852,015 disclose
stilbene derivatives having anti-viral effects.
[0015] U.S. Pat. No. 5,879,674 discloses methods of protecting
plant crops from insect attack by using stilbene derivatives to
induce epizootic viral infections.
[0016] U.S. Pat. No. 6,919,452 and US patent application
publication No. US 2005/0230662 disclose
4,4'-bis(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives and their use as fluorescent brightening agents.
[0017] Nowhere in the prior art is it taught or suggested that
stilbene derivatives may be used to treat aquatic mold
infections.
SUMMARY OF THE INVENTION
[0018] The present invention provides methods that are effective in
prevention and treatment of oomycete infections in aquatic
organisms. Advantageously, the methods of the invention involve use
of compositions comprising known compounds which have negligible
toxic effects and are acceptable for use even in aquatic species
intended for human consumption. In particular, the present
invention provides novel methods using stilbene derivatives for
treatment and prevention of oomycete infections. The invention is
based, in part, on the unexpected discovery that stilbene
derivatives, including
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives, are effective for prevention and treatment of oomycete
infections, such as saprolegniosis, which occur in aquatic
organisms including fish and fish eggs. The method of the invention
is effective for application at both an early stage of infection,
when no clinical signs may be apparent, as well as at later stages
of infection when infection is established. Without wishing to be
bound by any particular theory or mechanism of action, this
activity may be due to the ability of such compounds to interfere
with cell wall synthesis in oomycetes.
[0019] According to a first aspect, the present invention provides
a method of preventing or treating a oomycete infection in an
aquatic organism, the method comprising the step of contacting the
aquatic organism with an effective amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative of Formula (I):
##STR00001##
wherein R.sub.1 and R.sub.2 are the same or different and are each
independently selected from the group consisting of SO.sub.3H,
SO.sub.3Na, SO.sub.3K, SO.sub.3NH.sub.4 and H; and wherein R.sub.3
to R.sub.10 are the same or different and are selected from the
group consisting of H; linear or branched C.sub.1-C.sub.6 alkyl;
linear or branched C.sub.2-C.sub.6 alkenyl, wherein said alkyl or
alkenyl are each independently unsubstituted or substituted with a
hydroxyl, carboxyl, or carboxamide group; phenyl; and phenyl
substituted with R.sub.1 or R.sub.2 wherein R.sub.1 and R.sub.2 are
as defined above; or one or more of R.sub.3 and R.sub.4, R.sub.5
and R.sub.6, R.sub.7 and R.sub.9 or R.sub.5 and R.sub.10, together
with the nitrogen to which they are attached, form a heterocyclic
ring which can further comprise one or more additional heteroatoms
selected from N, O and S; and salts, hydrates, solvates and
polymorphs thereof.
[0020] According to another aspect, the present invention provides
a method of disinfecting equipment used for raising an aquatic
organism, wherein the equipment is contaminated with an oomycete,
the method comprising the step of contacting the equipment with an
effective amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative of Formula (I).
[0021] In particular embodiments of the methods of the invention,
two or more of R.sub.3, R.sub.4, R.sub.7 and R.sub.8 are the same
and are selected from the group consisting of CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CH.sub.2CONH.sub.2, CH.sub.3 and H;
or one or more of R.sub.3 and R.sub.4, R.sub.7 and R.sub.8 together
with the nitrogen to which they are attached, form a morpholinyl
ring. In other embodiments, two or more of R.sub.5, R.sub.6,
R.sub.9 and R.sub.10 are the same and are selected from the group
consisting of phenyl and phenyl substituted with SO.sub.3Na. In
other embodiments, R.sub.1 and R.sub.2 are the same and are
selected from SO.sub.3H and SO.sub.3Na. In still other embodiments,
R.sub.1 and R.sub.2 are different and are selected from SO.sub.3H,
SO.sub.3Na, SO.sub.3K, SO.sub.3NH.sub.4 and H.
[0022] In specific embodiments of the methods of the invention, the
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is selected from
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonic acid; [0023] disodium
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonate; [0024] potassium sodium
4,4'-bis-(6-anilino-4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino-
)stilbene-2,2'-disulfonate; [0025] 2,2'-stilbenedisulfonic acid,
4,4'-bis-(4-anilino-6-((2-hydroxyethyl)methylamino)-s-triazin-2-yl)amino)-
-, disodium salt; [0026] disodium
4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-d-
isulfonate; [0027] tetrasodium
4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulfonatoanilino)-1,3,5-triaz-
in-2-yl]amino]stilbene-2,2'-disulfonate; [0028] tetrasodium
4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,-
5-triazin-2-yl]amino]-stilbene-2,2'-disulfonate; [0029] and
2,2'-stilbenedisulfonic acid,
4,4'-bis-[[4-[(2-carbamoylethyl)(2-hydroxylethyl)amino]-6-(p-sulfoanilino-
)-s-triazin-2-yl]amino]-, tetrasodium salt.
[0030] In specific embodiments of the methods of the invention, the
oomycete is selected from Saprolegnia spp., Aphanomyces spp. and
Branchiomyces spp. In a more specific embodiment, the oomycete is
Saprolegnia parasitica.
[0031] In specific embodiments of the methods of the invention, the
at least one 4,4'-bis(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivative is provided as a solution. In other specific
embodiments, the at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is present in the solution at a concentration of about
20 to about 200 mg/L. According to preferred embodiments, the at
least one 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivative is present in the solution at a concentration of
about 25 mg/L.
[0032] In specific embodiments of the methods of the invention, the
aquatic organism is selected from fish, fish eggs and shellfish. In
specific embodiments, the fish are selected from barramundi, bass,
bream, carp, catfish, chub, eel, elver, flounder, gilthead, guppy,
halibut, koi, labrax, mullet, paddlefish, plaice, pompano, redfish,
red-drum, salmon, sole, sturgeon, tilapia, trout, tuna and
whitefish.
[0033] In specific embodiments of the methods of the invention, the
contacting step is for a period of about 2 to about 16 hours. In
preferred embodiments, the contacting step is for a period of about
8 hours. In other embodiments, the contacting step is repeated at
48 hour intervals.
[0034] In one embodiment of the method of preventing or treating an
oomycete infection in an aquatic organism, the oomycete infection
is concurrent with or accompanied by a parasitic infection. In
another embodiment, the parasitic infection is caused by at least
one parasite selected from the group consisting of Amyloodinium
spp., Argulus spp., Ascocotyle spp., Bothricephalus spp.,
Camallanus spp., Capilaria spp., Centrocestus spp., Chilodonella
spp., Coccidia spp., Contracaecum spp., Cryptobia spp.,
Cryptocaryon spp., Dactylogyrus spp., Dermocystidium spp.,
Ergasilus spp., Euclinostomum spp., Gyrodactylus spp., Hexamita
spp., Ichtyobodo spp., Ichtyophtirius spp., Lernaea spp.,
Metacercarius spp., Microsporidia spp., Myxosporea spp., Oodinium
spp., Sanguinicola spp., Sessiline spp., Spironucleus spp.,
Tetrahymena spp., Trichodina spp., Trichodinella spp. and
Tripartiella spp.
[0035] In one embodiment of the method of disinfecting equipment
used for raising an aquatic organism, the equipment is further
contaminated with at least one parasite. In another embodiment, the
at least one parasite is selected from the group consisting of
Amyloodinium spp., Argulus spp., Ascocotyle spp., Bothricephalus
spp., Camallanus spp., Capilaria spp., Centrocestus spp.,
Chilodonella spp., Coccidia spp., Contracaecum spp., Cryptobia
spp., Cryptocaryon spp., Dactylogyrus spp., Dermocystidium spp.,
Ergasilus spp., Euclinostomum spp., Gyrodactylus spp., Hexamita
spp., Ichtyobodo spp., Ichtyophtirius spp., Lernaea spp.,
Metacercarius spp., Microsporidia spp., Myxosporea spp., Oodinium
spp., Sanguinicola spp., Sessiline spp., Spironucleus spp.,
Tetrahymena spp., Trichodina spp., Trichodinella spp. and
Tripartiella spp.
[0036] It is to be understood explicitly that the scope of the
present invention encompasses variants of
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives, such as salts, hydrates, solvates and polymorphs
thereof, as are known in the art, with the stipulation that these
variants must preserve the capacity to prevent and treat oomycete
infections in aquatic organisms in the context of the present
invention.
[0037] Other objects, features and advantages of the present
invention will become clear from the following description and
drawings. However, it should be understood that the detailed
description and specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 illustrates the toxicity of various compounds in
tilapia fish. MG, Malachite Green; AmB, Amphotericin B
formulations; En, enilconazole; Det, detergents and disinfectants;
CS, copper sulfate; Py, Pyceze.RTM.; FA, formaldehyde; SPC, sodium
percarbonate; HP, hydrogen peroxide; BA, Blankophor.RTM. BA.
[0039] FIG. 2 illustrates the therapeutic efficacy of
Blankophor.RTM. BA (comprising potassium sodium
4,4'-bis-(6-anilino-4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino-
)stilbene-2,2'-disulfonate) and Malachite Green
(4-[(4-dimethylaminophenyl)phenyl-methyl]-N,N-dimethyl-aniline) in
a tilapia-saprolegniosis early infection model system. Tilapia fish
were treated with Blankophor.RTM. BA 100 mg/L (.diamond-solid.) or
200 mg/L ( ), or with Malachite Green 0.25 mg/L (.tangle-solidup.)
on the 2.sup.nd, 4.sup.th and 6.sup.th day after exposure to
Saprolegnia. Control, (.box-solid.).
[0040] FIG. 3 illustrates the therapeutic efficacy of
Blankophor.RTM. BA, formaldehyde and Malachite Green in a
tilapia-saprolegniosis early infection model system. Tilapia fish
were treated with Blankophor.RTM. BA 25 mg/L (.tangle-solidup.) or
50 mg/L (.diamond-solid.), Malachite Green 0.25 mg/L (.quadrature.)
or formaldehyde 50 mg/L ( ) on the 2.sup.nd, 4.sup.th and 6.sup.th
day after exposure to Saprolegnia. In additional experiments,
tilapia fish were treated with Blankophor.RTM. BA 50 mg/L on the
2.sup.nd and 9.sup.th day after exposure (.DELTA.), or 100 mg/L on
the 2.sup.nd day after exposure (.diamond.). Control,
(.box-solid.).
[0041] FIG. 4 illustrates the therapeutic efficacy of
Blankophor.RTM. BA, in a tilapia-saprolegniosis established
infection model system. Tilapia fish were treated with
Blankophor.RTM. BA 100 mg/L (.DELTA.), 50 mg/L (.smallcircle.) or
25 mg/L (.tangle-solidup.) on the 3.sup.rd and 8.sup.th day after
exposure to Saprolegnia. Control, (.diamond-solid.).
[0042] FIG. 5 shows scanning electron microscopy of cell walls of
Saprolegnia treated with (panel A) and without (panel B)
Blankophor.RTM. BA. Magnification: 3000.times..
[0043] FIG. 6 illustrates the effect of exposure to Blankophor.RTM.
BA (100 mg/L) (.quadrature.) on photosynthetic activity in pure
cultures of Chlorella (C), Microcystis (M), Peridinium (P) and
Melosira (Me), and in samples from Lake Kinneret (LK), as
determined by .sup.14C-uptake. Control, (.box-solid.).
DETAILED DESCRIPTION OF THE INVENTION
[0044] In accordance with the present invention, there are provided
methods for prevention and treatment of oomycete infections which
occur in aquatic organisms including fish and fish eggs, and
methods of disinfecting equipment used for raising aquatic
organisms, wherein the equipment is contaminated with an
oomycete.
[0045] The methods of the invention comprise a step of contacting
either the aquatic organism, or the equipment used for raising the
aquatic organism, with an effective amount of at least one stilbene
derivative, particularly a
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative of Formula (I):
##STR00002##
[0046] In Formula I, R.sub.1 and R.sub.2 are the same or different
and are each independently selected from the group consisting of
SO.sub.3H, SO.sub.3Na, SO.sub.3K, SO.sub.3NH.sub.4 and H. Further
in Formula I, R.sub.3 to R.sub.10 are the same or different, and
R.sub.3 to R.sub.10 are selected from the group consisting of H;
linear or branched C.sub.1-C.sub.6 alkyl; linear or branched
C.sub.2-C.sub.6 alkenyl, wherein said alkyl or alkenyl are each
independently unsubstituted or substituted with a hydroxyl,
carboxyl, or carboxamide group; phenyl; and phenyl substituted with
R.sub.1 or R.sub.2 wherein R.sub.1 and R.sub.2 are as defined
above; or, one or more of R.sub.3 and R.sub.4, R.sub.5 and R.sub.6,
R.sub.7 and R.sub.8 or R.sub.9 and R.sub.10, together with the
nitrogen to which they are attached, form a heterocyclic ring which
can further comprise one or more additional heteroatoms selected
from N, O and S.
[0047] In particular embodiments of the methods of the invention,
two or more of R.sub.3, R.sub.4, R.sub.7 and R.sub.8 are the same
and are selected from the group consisting of CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CH.sub.2CONH.sub.2, CH.sub.3 and H;
or one or more of R.sub.3 and R.sub.4, R.sub.7 and R.sub.8 together
with the nitrogen to which they are attached, form a morpholinyl
ring.
[0048] In other embodiments, two or more of R.sub.5, R.sub.6,
R.sub.9 and R.sub.10 are the same and are selected from the group
consisting of phenyl and phenyl substituted with SO.sub.3Na. In
other embodiments, R.sub.1 and R.sub.2 are the same and are
selected from SO.sub.3H and SO.sub.3Na. In still other embodiments,
R.sub.1 and R.sub.2 are different and are selected from SO.sub.3H,
SO.sub.3Na, SO.sub.3K, SO.sub.3NH.sub.4 and H.
[0049] Examples of
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives useful in the invention include, but are not limited to
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonic acid; disodium
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonate; potassium sodium
4,4'-bis-(6-anilino-4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino-
)stilbene-2,2'-disulfonate; 2,2'-stilbenedisulfonic acid,
4,4'-bis-(4-anilino-6-((2-hydroxyethyl)methylamino)-s-triazin-2-yl)amino)-
-, disodium salt; disodium
4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-d-
isulfonate; tetrasodium
4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulfonatoanilino)-1,3,5-triaz-
in-2-yl]amino]stilbene-2,2'-disulfonate; tetrasodium
4,4'-bis[[4-[bis(2-hydroxypropyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,-
5-triazin-2-yl]amino]-stilbene-2,2'-disulfonate; and
2,2'-stilbenedisulfonic acid,
4,4'-bis-[[4-[(2-carbamoylethyl)(2-hydroxylethyl)amino]-6-(p-sulfoanilino-
)-s-triazin-2-yl]amino]-, tetrasodium salt.
[0050] When the above-described compounds include one or more
chiral centers, the stereochemistry of such chiral centers can
independently be in the R or S configuration, or a mixture of the
two. The chiral centers can be further designated as R or S or R,S
or d,D, l,L or d,l, D,L.
DEFINITIONS
[0051] The term "C.sub.1 to C.sub.6 alkyl" as used herein refers to
saturated radicals such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, iso-butyl, sec-butyl, tert-butyl, amyl, tert-amyl, hexyl
and the like. Preferred C.sub.1 to C.sub.6 alkyl groups are methyl,
ethyl and propyl. The C.sub.1 to C.sub.6 alkyl groups are
optionally independently substituted with a hydroxyl, carboxyl or
carboxamide group. Preferred substituents are hydroxyl and
carboxamide groups. Exemplary substituted C.sub.1 to C.sub.6 alkyl
groups are hydroxyethyl, hydroxypropyl and ethylcarboxamido. In
specific embodiments of the
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives of the invention, each of R.sub.5, R.sub.6, R.sub.7 and
R.sub.8 are hydroxyethyl. In other specific embodiments, two of
R.sub.5 or R.sub.6, and R.sub.7 or R.sub.8 are hydroxyethyl and two
are H. In yet other embodiments, each of R.sub.5, R.sub.6, R.sub.7
and R.sub.5 are hydroxypropyl. In yet other embodiments, two of
R.sub.5 or R.sub.6, and R.sub.7 or R.sub.5 are ethylcarboxamido and
two are hydroxyethyl.
[0052] The term "C.sub.2 to C.sub.6 alkenyl" as used herein refers
to unsaturated radicals such as vinyl, allyl, 2-butenyl, 3-butenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl,
4-hexenyl, 5-hexenyl radicals attached at any appropriate carbon
position and the like, as well as dienes and trienes of straight
and branched chains. The C.sub.1 to C.sub.6 alkenyl groups are
optionally substituted with hydroxyl, carboxyl, or carboxamide
groups.
[0053] The 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives can be optionally substituted at any one or more
of R.sub.3 to R.sub.10 with phenyl groups or with phenyl groups
substituted with R.sub.1 or R.sub.2, wherein R.sub.1 and R.sub.2
are as defined above. In specific embodiments, two of R.sub.3 or
R.sub.4, and R.sub.9 or R.sub.10 are phenyl and two are H.
[0054] The term "phenyl substituted with R.sub.1 or R.sub.2" as
used herein refers to a phenyl group substituted with one or more
moieties chosen from the group consisting of SO.sub.3H, SO.sub.3Na,
SO.sub.3K, SO.sub.3NH.sub.4 and H. In specific embodiments, two of
R.sub.3 or R.sub.4, and R.sub.9 or R.sub.10 are phenyl substituted
with SO.sub.3Na and two are H.
[0055] The term "heterocyclic ring which can further comprise one
or more heteroatoms selected from N, O and S" as used herein refers
to optionally substituted five-membered to eight-membered rings
that can have 1 to 4 heteroatoms, such as nitrogen, oxygen and/or
sulfur, in particular oxygen, in conjunction with a nitrogen ring
atom. These five-membered to eight-membered rings may be saturated,
fully unsaturated, partially unsaturated or aromatic, with fully
saturated rings being preferred. The heterocyclic rings according
to the invention include, but are not limited to, morpholino,
piperidinyl, piperazinyl, imidazolyl, 2-amino-imidazoyl, pyrrolo,
heptylmethyleneimino, thiazole, triazole, tetrazole pyrrolidine,
pyrazole, imidazole, pyridine, thiomorpholine, oxazole and
pyrimidine.
[0056] The term "effective amount" as used herein refers to an
amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative of the invention which is effective to treat, prevent,
protect, repair, detoxify or disinfect an aquatic organism against
an oomycete infection or to treat, prevent, protect, repair,
detoxify or disinfect equipment against oomycete contamination,
upon contact with the at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative over some period of time.
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives
[0057] The 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives useful for the methods of the present invention
include known whitening agents which have been used in the textile,
detergent and paper industries. Such compounds are commonly known
as C.I. Fluorescent Brighteners (having various numerical
suffixes), and include, for example, C.I. Fluorescent Brightener
28, C.I. Fluorescent Brightener 113, C.I. Fluorescent Brightener
28/113, C.I. Fluorescent Brightener 220, C.I. Fluorescent
Brightener 235, C.I. Fluorescent Brightener 260 and C.I.
Fluorescent Brightener 263. C.I. Fluorescent Brighteners possess a
common backbone molecular structure, and differ with respect to the
substituents attached to either or both of the benzene and triazine
rings, and with respect to the nature of the salt or acid
derivative. Further, C.I. Fluorescent Brighteners are sold under
various trade names and one compound may have a number of different
synonyms. For example, disodium
4,4'-bis-(6-anilino-1,4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)ami-
no)stilbene-2,2'-disulfonate is known by the common name C.I.
Fluorescent Brightener 28 and has been sold under the trade names
Calcofluor.RTM. White, Cellufluor.RTM. and Phorwite.RTM., among
others. C.I. Fluorescent Brighteners are supplied as aqueous
solution or powder formulations. For example, Blankophore.RTM. BA
Liquid (Lanxess) is an aqueous solution comprising the mixed salt
potassium sodium
4,4'-bis-(6-anilino-4-bis)-2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino-
)stilbene-2,2'-disulfonate.
[0058] Other
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives are also envisioned, and can be created by changing the
substituents attached to either or both of the benzene and triazine
rings, and/or the salt derivative. They may be in the form of
salts, mixed salts, free acids and mixtures thereof. Such
derivatives can be readily designed and synthesized by those of
skill in the art.
[0059] The 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives useful for the methods of the present invention
may be provided in various formulations, for example, aqueous
solutions, or powder formulations which are reconstituted with
water. Aqueous formulations may contain agents which aid in
solubility. Powder formulations may contain dispersing agents or
dedusting agents. Such excipients should be appreciably soluble in
water and be non-toxic to living organisms and the environment.
[0060] The 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives useful for the methods of the present invention
are appreciably soluble in water. The water solubility can in
general, be increased by increasing the numbers of hydrophilic
substituents such as sulfonate and hydroxyl groups, as is known to
those skilled in the art.
[0061] The 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives useful for the methods of the present invention
are stable to hydrolysis, but may be subject to photodegradation in
the hydrosphere, due to having UV absorption maxima between 340 to
360 nm in water. They may or may not be readily biodegradable; in
the latter case, they are preferably adsorbed onto sludge in
wastewater treatment systems.
[0062] The 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives useful for the methods of the present invention
have minimal impact on living creatures and on the environment. For
example, the overall relative safety of C.I. Fluorescent
Brighteners 28, 220, 235, 260 and 263 has been documented. These
compounds are reported to have low toxicity to fish, annelids and
bacteria and low to moderate toxicity to aquatic invertebrates and
algae. With respect to mammals, they have low acute or repeated
dose oral toxicity, are not mutagenic or clastogenic, are not
reproductive or developmental toxicants, and are generally not
irritating or sensitizing to skin and eyes (Stilbene Fluorescent
Whitening Agents Category, submitted to the US Environmental
Protection Agency by the ETAD Fluorescent Whitening Agent Task
Force, Oct. 6, 2005).
Putative Mechanism of Action
[0063] Without wishing to be bound to any particular theory or
mechanism of action, the therapeutic and prophylactic efficacy of
the 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives of the invention against oomycetes may be attributed to
the ability of such compounds to disrupt oomycete cell walls.
Various 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives are known to bind to polysaccharides having
.beta.-glucosidic linkages, such as occur in cellulose (a
.beta.-(1,4) linked polymer of D-glucose units). Oomycete cell
walls in general, contain cellulose and other .beta.-glucan
polymers, for example .beta.-(1,2) linked and (.beta.-(1,6) linked
polymers of D-glucose units, as the major structural elements.
While the proportions and linkages of these polymers vary among
different oomycete genera and species, these macromolecules are the
likely targets of the
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives of the invention. Intercalation of such compounds with
fully formed and/or nascent .beta.-glucosidic polymers compromises
the integrity of the cell wall, ultimately leading to cell
lysis.
[0064] The mechanism of action may be similar or analogous to that
exerted by 4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic
acid derivatives against true fungi, in which chitin (a
.beta.-(1,4) linked polymer of N-acetyl-D-glucosamine units) is the
major structural component of cell walls.
Oomycete Pathogens and Diseases of Aquatic Organisms
[0065] The methods of the invention are effective against disease
causing oomycetes which attack aquatic organisms, including fish
and shellfish. Oomycetes include but are not limited to Achyla
spp., Aphanomyces spp., Branchiomyces spp., Brevilegnia spp.,
Dermocystidium spp., Dictyuchus spp., Ichthyophonus spp., Isoachyla
spp., Leptolegnia spp., Leptomitus spp., Protoachyla spp., Pythium
spp., Saprolegnia spp., and Thraustotheca spp.
[0066] Organisms of the genus Saprolegnia, and especially S.
parasitica, are responsible for saprolegniosis, one of the most
common and significant oomycete infections occurring in fish. In
common with all saprophytic oomycetes, Saprolegnia spp. feeds by
secreting degradative enzymes onto the surface to which it is
attached, thus enabling absorption of nutrients such as proteins
and carbohydrates. Saprolegniosis often occurs as a secondary
infection following damage to the fish integument (skin and gills)
caused by parasites, viruses, bacterial infections and abrasion.
Other predisposing factors include overcrowding, intensive handling
and water pollution. Less commonly, Saprolegnia spp. can act as a
primary pathogen infecting fish which have not incurred integument
damage. Such attacks are temperature-dependant, usually occurring
at low temperatures, possibly as a consequence of a reduced immune
response. As well as being a threat to fish, Saprolegnia spp. also
infects fish eggs.
[0067] Saprolegniosis appears as grey/white patches on the skin or
gills that resemble tufts of cotton wool. At a later stage they may
become brown or green as they trap sediment or algae. If the fish
is removed from the water, the fungus appears as a slimy matted
mass. Saprolegnia spp. normally establishes as small, focal
infections that then spread rapidly over the body or gills. As it
spreads, healthy tissue is destroyed. There is often little
inflammation unless there is an underlying bacterial infection.
Microscopic examination shows broad, non-septate hyphae, typical of
oomyetes.
[0068] Achyla spp. cause a disease similar to saprolegniosis, and
under similar conditions.
[0069] Branchiomycosis or "gill rot" is caused by Branchiomyces
sanguinis and B. demigrans. Both species are found in fish
suffering from environmental stress, such as low pH, low dissolved
oxygen, high algal bloom or high ammonia levels. Affected fish
appear lethargic and the gills are striated or marbled with the
pale areas representing infected and dying tissue.
[0070] Oomycete infections are transmitted among fish by infectious
zoospores which are released from infected tissues.
Aquatic Organisms
[0071] According to the invention, the method of preventing or
treating an oomycete infection can be applied to a wide variety of
aquatic organisms that are infected with or at risk of infection by
oomycete pathogens. Aquatic organisms include but are not limited
to fish and eggs thereof. The fish include economically useful fish
raised in commercial fish farming settings, cultured fish, aquarium
fish and decorative fish of all ages which live in fresh water and
sea water. The fish include but are not limited to barramundi,
bass, bream, carp, catfish, chub, eel, elver, flounder, gilthead,
guppy, halibut, koi, labrax, mullet, paddlefish, plaice, pompano,
redfish, red-drum, salmon, sole, sturgeon, tilapia, trout, tuna and
whitefish. Aquatic organisms further include but are not limited to
shellfish, both molluscs and crustaceans, for example abalone,
clam, crab, geoduck, mussel, lobster, oyster, prawn, shrimp and
urchin.
Equipment Used for Raising Aquatic Organisms
[0072] According to the invention, the method of disinfecting
equipment used for raising aquatic organisms is directed to
equipment that is used for containing, raising, manipulating and
treating the aquatic organisms, for example fish. Detection of
oomycete disease in the aquatic organisms is a sufficient indicator
that the equipment is contaminated, either by direct contact with
the infected aquatic organisms harboring hyphal mycelia or by
contact with the dispersed zoospores. Such equipment requires
disinfection, so as to eliminate the possibility of recurrent
infection, for example, in mature fish that have had an oomycete
disease and have been treated according to the invention, or new
infection in juvenile fish or eggs introduced to or exposed to the
equipment. The equipment includes aquariums, basins, baths, cages,
filters, meshes, nets, ponds, pools, tanks, transfer apparatus,
troughs, thermometers and the like.
Parasitic Infections
[0073] Parasitic infections are often concurrent with or accompany
oomycete infections, for example in commercial fish farming
settings and in hobby aquariums. Fish subjected to stress and/or
adverse environmental conditions may be vulnerable to such
concurrent infections due to immune system suppression. Parasitic
infections include those caused by Amyloodinium spp., Argulus spp.,
Ascocotyle spp., Bothricephalus spp., Camallanus spp., Capilaria
spp., Centrocestus spp., Chilodonella spp., Coccidia spp.,
Contracaecum spp., Cryptobia spp., Cryptocaryon spp., Dactylogyrus
spp., Dermocystidium spp., Ergasilus spp., Euclinostomum spp.,
Gyrodactylus spp., Hexamita spp., Ichtyobodo spp., Ichtyophtirius
spp., Lernaea spp., Metacercarius spp., Microsporidia spp.,
Myxosporea spp., Oodinium spp., Sanguinicola spp., Sessiline spp.,
Spironucleus spp., Tetrahymena spp., Trichodina spp., Trichodinella
spp. and Tripartiella spp.
[0074] The inventors of the present invention have surprisingly
found that treatment with at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative according to the invention is effective for treating at
least one parasitic infection which is concurrent with an oomycete
infection in fish.
Applications of the Invention
[0075] The method of preventing or treating an oomycete infection
described herein can be applied in a variety of situations,
including: (1) prophylactic prevention of seasonal disease
outbreaks in commercial fish farming settings; (2) intervention and
therapeutic treatment of infected fish; (3) treatment prior to
anticipated stress conditions, for example changes in water quality
including temperature decrease; (4) pretreatment and treatment
after transferring and/or shipping fish; (5) treatment of "sick"
fish for home hobbyists; and (6) maintenance of health in fish for
small and large scale, domestic and commercial growth and for
scientific experiments. The method of the invention is effective
for application at both an early stage of infection, when no
clinical signs may be apparent, as well as at later stages of
infection when the oomycete infection is established and causes a
high rate of mortality.
[0076] For preventing or treating an oomycete infection, aquatic
organisms are contacted with an effective amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative. The contacting step may be carried out by transferring
aquatic organisms, for example fish or shellfish in a commercial
setting, from their original containment facility, for example a
pond or tank, to a fresh containment facility containing an
effective amount of at least one
4,4'-bis(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative. This method is preferable when oomycete infection has
been detected, for example, on the bodies of fish, requiring
appropriate intervention and therapeutic treatment. For such
therapeutic treatment, the density of fish in the fresh containment
facility may be maintained at the same ratio as that in the
original containment facility, or may be preferably decreased to
ease stress conditions.
[0077] Alternately, the organisms may be maintained in their
original containment facility, to which is added an effective
amount of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative. This method may be preferred for economic reasons when
the method of the invention is applied for prevention of oomycete
infection, either as a routine procedure or in anticipation of
expected stress conditions.
[0078] In the methods described herein for preventing or treating
an oomycete infection and for disinfecting equipment, the
contacting step is carried out with at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative which may be provided in a variety of forms. A solution
is generally preferred, but other forms are also envisioned, for
example, dissolving tablets, gels, and impregnated materials which
release the active material upon exposure to water, or which are
suitable for direct application to contaminated equipment.
[0079] When a solution is used, a concentration in the range of
about 20 to about 200 mg/liter of water may be suitable for most
applications. The lowest concentration which achieves preventative,
therapeutic, or disinfecting efficacy against oomycete infection or
contamination is preferable, in order to minimize costs and the
amount of compounds released into waste water systems. The
preferred concentration may further depend on duration of the
treatment and on the age and condition of the treated aquatic
organisms. For example, contacting of tilapia fish with 25 mg/liter
of Blankophor.RTM. BA for 8 hours has been found to be effective
for preventing and treating saprolegniosis in tilapia fish.
[0080] An effective amount of the at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is that which achieves preventative (prophylactic),
therapeutic, or disinfecting efficacy, as appropriate. The
effective amount may be determined in pilot experiments. An
effective amount for preventing oomycete infection refers to the
amount or concentration brought into contact with an aquatic
organism such that the aquatic organism is prevented from becoming
infected in the presence of an oomycete pathogen to which the
aquatic organism is susceptible. For example, a prophylactic
treatment is deemed to be effective in a situation where the
mortality rate due to oomycete pathogen challenge in fish
pretreated with a
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative prior to challenge is reduced by a significant
percentage, for example, 25 to 100%, of the rate observed in fish
not pretreated. An effective amount for treating an oomycete
infection refers to the amount or concentration brought into
contact with an aquatic organism infected with an oomycete pathogen
such that the aquatic organism is protected against the development
or progression of an infection, disease, or mortality associated
with the oomycete pathogen. For example, a therapeutic treatment is
deemed to be effective in a situation where the mortality rate in
fish first challenged with an oomycete pathogen and subsequently
treated with a
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative is reduced by a significant percentage, for example, 25
to 100%, of the rate observed in infected, non-treated fish. An
effective amount for disinfecting equipment contaminated with an
oomycete refers to the amount or concentration brought into contact
with the equipment such that the equipment no longer harbors
oomycete mycelia or zoospores. For example, a disinfecting
treatment is deemed to be effective in a situation where the
disinfected equipment is monitored for the presence of oomycete
mycelia and/or zoospores after the disinfecting treatment and found
to be reduced by a significant percentage, for example, 25 to 100%,
of that observed before the disinfecting treatment.
[0081] Contacting of aquatic organisms and/or contaminated
equipment with at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative may be carried out over a period of about 2 hours to
about 16 hours. For example, a period of about eight hours has been
found to be effective in infected fish. If time is a limitation,
relatively shorter periods may be used, optionally with higher
concentrations of at least one
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivative. If time is not a limiting factor, longer periods may be
used and may be preferable for disinfecting equipment. Further, the
contacting periods may be repeated at regular intervals, for
example, 48 hours after completion of the first contacting period,
to ensure complete eradication of the oomycete infection or
contamination.
[0082] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the brand concept, and,
therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a
variety of alternative forms without departing from the
invention.
[0083] The following examples are presented in order to more fully
illustrate certain embodiments of the invention. They should in no
way, however, be construed as limiting the broad scope of the
invention. One skilled in the art can readily devise many
variations and modifications of the principles disclosed herein
without departing from the scope of the invention.
EXAMPLES
Example 1
Susceptibility Testing of Saprolegnia parasitica
[0084] The in vitro susceptibility of Saprolegnia parasitica T-1
and S. parasitica CBS 540.67 to various compounds was determined by
the agar-dilution and broth macrodilution methods.
Material and Methods
[0085] Saprolegnia parasitica Isolates
[0086] Saprolegnia parasitica T-1 isolate was obtained from skin
lesions on moribund tilapia exhibiting signs of saprolegniosis. For
the purpose of isolation, a small part of the skin and scales
covered with hyphae was dried, and cut with a sterile scalpel
blade, then placed onto Glucose-Peptone-Penicillin-Streptomycin
(GP-PS) plates containing 3 g/l glucose, 1 g/l peptone, trace
micronutrients and 250 mg/l each Penicillin-G and Streptomycin
Sulphate, and incubated at 18.degree. C.
[0087] After incubation for 3-4 days, a block of agar from the edge
of the fungal colony was cut off and placed in sterile Petri
plates, containing double distilled water and sterile Clover seeds,
in order to obtain new bacteria-free colonies. The molecular
identification was done by Dr. Alexandra Riethmueller (University
of Kassel, Germany) by sequencing of the ITS regions of the 18S
rDNA.
[0088] The isolates were kept at 18.degree. C. on GP-PS plates, and
were transferred every month.
S. parasitica CBS 540.67 is a publicly available strain.
[0089] Reagents
[0090] Compounds tested for activity against S. parasitica are
summarized in Table 1.
[0091] Susceptibility Testing
[0092] Two methods were used, each suitable for a different phase
of the asexual life cycle of Saprolegnia spp.: i) the broth
macrodilution method, for determination of susceptibility of
zoospores and cysts; and ii) the agar-dilution method for
determination of susceptibility of filamentous hyphae. Each
compound was tested three times in each method. Malachite Green was
used as positive control.
[0093] Broth Macrodilution Method
[0094] This method is a modification of the method used for molds
according to EUCAST 7.1 (European Committee on Antibiotic
Susceptibility Testing) and CLSI/NCCLS M27-A2 (Clinical Laboratory
Standards Institute/National Committee for Clinical Laboratory
Standards). For inoculum preparation, S. parasitica isolates were
cultured on GP-PS for 7 days with sterile clover seeds at
18.degree. C. Clover seeds (.about.20) covered with mycelium were
removed and added to 40 ml of RPMI-1640 broth medium (Sigma;
prepared according to NCCLS instructions) and incubated at
18.degree. C. for 4-7 days. Cysts and zoospores free from hyphae
were collected, and an inoculum of .about.10.sup.3 CFU/ml as
measured by hemacytometer counting, was prepared in RPMI. Stock
solutions of the tested compounds were prepared in water or in
DMSO, as indicated in Table 2. Fluorescent Brightener 28 stock
solution was prepared by adding 40% KOH gradually to a 10 mg/ml
solution in water, until it was clear, then it was filtered twice
through a 0.2 .mu.m cellulose acetate nonpyrogenic sterile filter
(Schleicher & Schuell, Dassel, Germany).
[0095] Twofold serial dilutions of the resulting stock solutions
were set in RPMI-1640 medium in a volume of 0.1 ml. Final drug
concentrations in the test tubes ranged from 1.times.10.sup.4 to
0.01 mg/L. Inoculum of 0.9 ml was added to each of a sterile tube
containing 0.1 ml of the serially diluted drug. Two tubes
containing drug-free medium and two tubes containing drug-free
inoculum were used as controls. The inoculated tubes were incubated
at room temperature (15-20.degree. C.) for 24 h. The growth in each
tube was then evaluated visually. The MIC-0 was defined as the
lowest drug concentration that resulted in complete inhibition of
visible growth.
[0096] Agar-Dilution Method
[0097] This method is a modification of the method presented by
Bailey (Bailey, T. A. (1983) Prog. Fish Cult. 45:24-27), and was
used for the susceptibility testing of Saprolegnia hyphae. Twofold
serial dilutions were prepared in double distillated sterile water
with final drug concentrations ranging from 10,000 to 0.01 mg/L in
a final volume of 1.0 ml. Melted (56.degree. C.) RPMI-1640 agar
medium (Sigma) was prepared according to NCCLS instructions, and
then added to each tube containing drug or water control. The
mixture was vortexed, and poured into small plates. After agar
solidification a small measured piece, taken from a 7-10 day old
culture of S. parasitica T-1 on GP-PS medium was placed upside down
on each plate. The plates were incubated at room temperature
(15-20.degree. C.) for 24 h. The growth in each plate was then
estimated visually, and the MIC values were defined as the minimal
concentration that resulted in complete inhibition of visible
growth compared to a drug-lacking control.
TABLE-US-00001 TABLE 1 Compounds tested against S. parasitica
Compound Source Malachite Green Reactif R.A.L, Paris, France Copper
sulfate Sigma, St. Louis, Mo., USA Formaldehyde Sigma, St. Louis,
Mo., USA Fluorescent Brightener 28 Sigma, St. Louis, Mo., USA
Pyceze .RTM. (bronopol) Novartis, Essex, UK Blankophore .RTM. BA
Lanxess, Leverkusen, Germany Hydrogen peroxide Ecolab, Zohar Dalia,
Israel Sodium percarbonate Ecolab, Zohar Dalia, Israel Clinador
.RTM. (Enilconazole) Dorvet, Nes Ziona, Israel Amphotericin B
Dumex, Copenhagen, Denmark Fungizone .RTM. (amphotericin Squibb,
Middlessex, UK deoxycholate) Amphotericin B- Falk, R., Domb, A. J.
& arabinogalactan conjugate Polacheck, I. (1999) (AmB-AG)
Antimicrobial Agents and Chemotherapy 43, 1975-81 C-TAB BDH
Chemicals, Poole, UK Triton X-100 BDH Chemicals, Poole, UK Tween
.RTM.-20 BDH Chemicals, Poole, UK Tween .RTM.-80 BDH Chemicals,
Poole, UK Sodium dodecylsulfate (SDS) BDH Chemicals, Poole, UK
Digitonin Merck, Dermstadt, Germany Carcid .RTM. Carmel Resins,
Atlit, Israel Carcil .RTM. C-50 Carmel Resins, Atlit, Israel
Septocil .RTM. Carmel Resins, Atlit, Israel Agrosept .RTM. Ketox,
Copenhagen, Denmark Neem oil Tomer-teva, Israel Saprofin .RTM.
Dropco Laboratorios, Chile NaCl Sigma, St. Louis, Mo., USA
Results
[0098] The two methods for determining in vitro susceptibility of
S. parasitica were highly reproducible (98 and 95% respectively).
The MIC values obtained using the two S. parasitica isolates were
similar for each compound tested, and the difference between them
was statistically insignificant. The MIC values of the tested
compounds are presented in Table 2, with each result representing
the geometric mean of three independent tests. The lowest MIC
values were consistently obtained with Malachite Green (0.06 mg/L).
Other compounds that exhibited high in vitro activity
(MIC.ltoreq.10 mg/L) against S. parasitica were as follows:
Fluorescent Brightener 28, all the tested cationic detergents,
Digitonin (a non-ionic detergent), sodium percarbonate and hydrogen
peroxide. The lowest activity was detected with natural oils,
Tween-20 and sodium chloride (MIC.gtoreq.200 mg/L). Despite the
high in vitro activity observed with Fluorescent Brightener 28 (MIC
values of 1 and 5 mg/L in both methods), it was eliminated from
subsequent studies on in vivo activity, toxicity and therapeutic
efficacy, due to its extremely high cost.
TABLE-US-00002 TABLE 2 MIC values (mg/L) of various compounds S.
parasitica T-1 S. parasitica CBS 540.67 Agar- Agar- Compound
Solvent Macrodilution dilution Macrodilution dilution
Antimicrobials Malachite Green DDW 0.06 0.06 0.06 0.06 Sodium
percarbonate DDW 5 NA* 2.5 NA Hydrogen peroxide DDW 10 NA 8 NA
Copper sulfate DDW 40 80 80 120 Formaldehyde DDW 100 100 100 100
Pyceze .RTM. (bronopol) DDW 100 100 100 200 Sodium chloride DDW
30,000 NA 30,000 NA Antifungal agents Fungizone .RTM. DDW 0.25 5
0.5 5 Amphotericin B DMSO 0.5 5 0.25 2.5 AmB-AG conjugate DDW 0.5 5
0.25 5 Enilconazole DDW 1 5 1 5 Fluorescent Brighteners Fluorescent
DDW 1 5 2 5 Brightener 28 Blankophore .RTM. BA DDW 100 200 200 200
Detergents & disinfectants Carcid .RTM. (cationic) DMSO 5 50 5
100 Carcil .RTM. C-50 DMSO 5 50 5 50 (cationic) Septocil .RTM.
(cationic) DMSO 5 50 5 100 CTAB (cationic) DMSO 5 10 5 50 Agrosept
.RTM. (cationic) DMSO 10 50 5 50 Digitonin (non ionic) DMSO 2.5 10
2.5 20 Triton X-100 DMSO 50 200 100 200 (non ionic) Tween .RTM. 20
DMSO 500 1,000 250 1,000 (non ionic) Tween .RTM. 80 DMSO 100 200
100 200 (non ionic) SDS (anionic) DMSO 100 500 250 500 Natural oils
Saprofin .RTM. DMSO 400 400 400 400 Neem oil DMSO >1,000
>1,000 >1,000 >1,000 NA = Non-applicable
Example 2
Toxicity Testing in Tilapia
[0099] Compounds evaluated in Example 1 were further evaluated for
their toxic effects in tilapia fish.
Material and Methods
[0100] LC.sub.50 values were determined by static short-term tests
as a measure of the relative acute lethal toxicity for tilapia
(Standard methods for the evaluation of water and wastewater (1985)
16th ed. American Public Health Association, Washington, D.C. p.
689-819).
[0101] Hybrid tilapia (Oreochromis niloticus X Oreochromis aureus)
having an average weight of 20 g, were kept in 100 liter
polyethylene tanks at a density of 1 fish per 10 liters, at a
constant temperature of 21.degree. C. for three weeks before the
initiation of the experiment by addition of a test compound to each
tank. The toxicities of hydrogen peroxide, sodium percabonate and
Blankophore.RTM. BA were also evaluated at temperatures of
18.degree. C. and 15.degree. C. For the latter experiments, fish
were acclimated to the required temperature for three weeks prior
to addition of the test compound to the tank. Fish were treated
with 5 different concentrations of each test compound, determined
according to the MIC values of each compound.
[0102] Mortality of fish was recorded up to 96 h. Water parameters
(O.sub.2, NH.sub.4.sup.+, NO.sub.2.sup.-, pH and Cl.sup.-) were
monitored throughout the experiment and were maintained within
acceptable limits. The presented results are the mean of two
separate experiments.
[0103] All procedures for care and treatment of fish were in
accordance with the Guide for the Care and Use of Laboratory
Animals (Hebrew University of Jerusalem, Israel) and were approved
by the Committee for Ethical Conduct in the Care and Use of
Laboratory Animals.
Results
[0104] LC.sub.50 values determined for various compounds are
presented in FIG. 1. Compounds having an LC.sub.50 value 5 to 10
times higher than that of their respective MIC were selected for
further study for therapeutic efficacy in the
tilapia-saprolegniosis model.
[0105] The LC.sub.50 values of copper sulfate, Pyceze.RTM., and all
the tested disinfectants, detergents and antifungal agents were
lower, similar or insignificantly different from their respective
MIC values and were accordingly excluded from the therapeutic
efficacy study. Significantly higher differences were demonstrated
for Malachite Green, formaldehyde, hydrogen peroxide, sodium
percarbonate, and Blankophore.RTM. BA i.e. the ratio between the
LC.sub.50 value and the MIC value was >20. Some of the compounds
with low toxicity at 18.degree. C. were also tested at a lower
temperature (15.degree. C.). The results are presented in Table 3
and clearly indicate that the toxicity of hydrogen peroxide, and to
a greater extent, sodium percarbonate, increased significantly at
15.degree. C. In contrast, the LC.sub.50 value of Blankophor.RTM.
BA remained high, even at the lower temperature, indicating its
relative lack of toxicity for the fish.
TABLE-US-00003 TABLE 3 Toxicity of compounds (LC.sub.50) in tilapia
at various temperatures LC.sub.50 (mg/L) Compound 15.degree. C.
18.degree. C. 25.degree. C. Hydrogen peroxide 75 .gtoreq.100
>100 Sodium percarbonate 27 >100 >100 Blankophor .RTM. BA
>2,000 >2,000 >2,000
Example 3
Toxicity Testing of Blankophor.RTM. BA in Various Fish Species
Materials and Methods
[0106] LC.sub.50 values for Blankophor.RTM. BA in various fish
species were determined as in Example 2, except that fish were
acclimated to 21.degree. C. for three weeks, and then to 15.degree.
C. for another two weeks before the initiation of the experiment.
Following acclimation to 15.degree. C., Blankophor.RTM. BA (100,
200, 500, 1,000 or 2,000 mg/L) was added to the tanks, and
mortality of fish was recorded up to 96 h. Water parameters
(O.sub.2, NH.sub.4.sup.+, NO.sub.2.sup.-, pH and Cl.sup.-) were
monitored throughout the experiment and were maintained within
acceptable limits. The presented results are the mean of two
separate experiments. The fish used were: hybrid tilapia
(Oreochromis niloticus X O. aureus), average weight 20 g; grey
mullet (Mugil cephalus), average weight 70 g; common carp (Cyprinus
carpio), average weight 25 g; hybrid striped bass (Morone saxatilis
X M. chrysops), average weight 25 g, and grass carp
(Ctenopharyngodon idella), average weight 25 g.
Results
[0107] The LC.sub.50 values observed for the fish species tested
were all greater than 2000 mg/L. These results are consistent with
the results presented in Example 2 and support the conclusion that
compounds of the invention, such as Blankophor.RTM. BA, are
substantially non-toxic to a variety of fish species, including
those of commercial importance.
Example 4
Therapeutic Efficacy of Blankophor.RTM. BA in an Early Infection
Model of Saprolegniosis
Material and Methods
[0108] Hybrid tilapia (Oreochromis niloticus X Oreochromis aureus),
with an average weight of 20 g were treated against ectoparasites
using 25 .mu.g/ml of formaldehyde (37% v/v) two weeks prior to the
experiment, and were maintained at a constant temperature of
21.degree. C. in a 100 liter polyethylene tank. The water tank was
cooled from an initial temperature of 21.degree. C. to 14.degree.
C. over a period of 10 days. The water was kept at a constant
temperature of 14.degree. C. and the fish were acclimated for 4
days. Physical stress of abrasion was then applied by agitating 40
fish for 10 seconds within a plastic net (30.times.20 cm).
Following this, a sterile stainless steel tea infuser containing 60
clover-seeds covered with hyphae of Saprolegnia parasitica T-1 (7
day old culture on GP-PS medium at 18.degree. C.) was added to each
tank, in which the fish density was 20 fish per 100 liters. This
treatment lasted 48 h and allowed the dispersion of zoospores that
were produced. The density of the zoospores in the water was
determined according to the method described below, and was
estimated as 5.times.10.sup.2-2.times.10.sup.3 zoospores per liter.
The fish were then transferred to different tanks, and treated with
Blankophor.RTM. BA (200, 100, 50 or 25 mg/L) or Malachite Green
(0.25 mg/L) for 8 h at 13-14.degree. C., with 10 fish per 100 L
water. The treatments were applied on 2.sup.nd, 4.sup.th and
6.sup.th day following initiation of the experiment. The fish were
monitored for 14 days for the presence of any lesion covered with
hyphae, indicative of Saprolegnia infection. Skin biopsies of
diseased and moribund fish were checked microscopically (10.times.
and 40.times. magnification). Morbidity and mortality was monitored
daily. Moribund and or dead fish were removed from all tanks during
the experimental period.
[0109] The determination of zoospore density in the water was
performed according to Willoughby, L. G. (1994) Fungi and fish
diseases, Pisces Press, Stirling, Scotland p. 57.
[0110] In brief, a 1 liter sample of water from the 100 liter tank
was divided into 1 and 10 ml aliquots in sterile Petri dishes. Each
sample was diluted with water to give a final volume of 20 ml and
then one sterile clover seed was added in order to attract and keep
the zoospores. All the water samples were incubated at room
temperature for 72 h allowing spore germination. The density of the
zoospores was determined according to the number of seeds covered
with visible mycelium.
Results
[0111] Saprolegnia-infected fish were treated with Blankophor.RTM.
BA at different concentrations (100 and 200 mg/L), in accordance
with the determined MIC values. Treatments were carried out on the
2.sup.nd, 4.sup.th and 6.sup.th days after the initiation of the
infection, and each treatment was for a period of 8 h. Fish treated
with 0.25 mg/L Malachite Green served as a positive treatment
control. Fish were monitored for 14 days. All dead fish were
clinically evaluated, and exhibited significant Saprolegnia
lesions, as determined microscopically and confirmed by positive
culture of Saprolegnia. The results of the treatment with
Blankophor.RTM. BA are summarized in FIG. 2, and show that
Blankophor.RTM. BA is as effective as Malachite Green in enabling
survival of fish following Saprolegnia infection. All fish treated
with Blankophor BA survived for at least 10 days following
initiation of the infection, in contrast to untreated fish which
progressively died from the 3.sup.rd day.
[0112] Due to the high efficacy of the treatment with 100 mg/L
Blankophor.RTM. BA, additional experiments were performed using
lower concentrations of Blankophor.RTM. BA (50 and 25 mg/L). As in
the previous experiments, infected fish were treated in tanks
containing different concentrations of the compound on the
2.sup.nd, 4.sup.th and 6.sup.th days after the initiation of the
infection. In addition, two additional treatment regimens were
added i.e. 50 mg/L (administered on the 2.sup.nd and 9.sup.th days
after initiation of the infection) and 100 mg/L (administered on
the 2.sup.nd day after initiation of the infection). The results
are summarized in FIG. 3 and indicate that Blankophor.RTM. BA was
highly effective in preventing and treating early stage
saprolegniosis infection in tilapia, even at a concentration as low
as 25 mg/L.
[0113] Clinical observation of fish treated with high dosages (50
and 100 mg/L) of Blankophor.RTM. BA indicated that the specimens
were free of lesions (a typical clinical sign) and furthermore,
were negative upon culture for Saprolegnia. In addition, biopsies
of scale and fin material indicated significant differences in the
Saprolegnia hyphae removed from fish treated with Blankophor.RTM.
BA 25 (mg/L) versus that removed from non-treated fish.
Specifically, the former exhibited "normal" Saprolegnia hyphal
structure, while the latter exhibited hyphae which were severely
damaged, and considerably slimmer.
[0114] Formalin (37% formaldehyde v/v) was also tested in the
tilapia-saprolegniosis model. At a concentration of 100 mg/L
(administered on the 2nd, 4th and 6.sup.th days after the
initiation of the infection) 70% of the fish died within 14 days
(data not shown). At a concentration of 50 mg/L, the therapeutic
efficacy was observed to be similar to that of Blankophor.RTM. BA
(FIG. 3). Formalin is not a feasible treatment for saprolegniosis
however, since formaldehyde has a number of serious deleterious
effects on animals and the environment. For example, it is a
neurotoxin and probable carcinogen for humans; it decreases the
soluble oxygen concentration in water, and is an algaecide.
Example 5
Therapeutic Efficacy of Blankophor.RTM. BA in an Established
Infection Model of Saprolegniosis
Materials and Methods
[0115] Tilapia were exposed to S. parasitica and treated with
Blankophor.RTM. BA (100, 50 and 25 mg/L), as described in Example
4, with the exception that Blankophor.RTM. BA treatment was started
on the 3.sup.rd day after initiation of the infection, and repeated
on the 8.sup.th day. By the starting point of the Blankophor.RTM.
BA treatment, approximately 50% of the fish had established
infection, as indicated by the presence of white skin lesions and a
mortality rate of approximately 30%.
Results
[0116] FIG. 4 shows that in this model of established
saprolegniosis, treatment with Blankophor.RTM. BA (50 and 100 mg/L)
was highly effective in promoting survival of infected fish. The
difference between the non-treated control and Blankophor-treated
fishes was highly significant (p<0.01).
Example 6
Non-Randomized Field-Trial of Blankophor.RTM. BA Treatment for
Concurrent Oomycete and Parasitic Diseases
[0117] Following transfer of 2000 hybrid tilapia fish (20-30 g) to
a cement pond (1.times.10.sup.5 L, 21 to 25.degree. C.), white
patches of filamentous mycelium were observed on the body and fins
of fish, covering in most cases about 80% of the body surface. The
mycelia appeared about four days after the transfer, presumably due
to opportunistic infection by Saprolegnia sp. following handling
stress. Death of 20 to 30 fish per day was recorded. All the dead
fish were covered with mycelium, characteristic of Saprolegnia sp.,
and were also found to be infected with the parasites Gyrodactilus
sp. and Trichodina sp.
[0118] The water capacity in the pond was decreased to
3.times.10.sup.4 L, and treatment with Blankophor.RTM. BA (50 mg/L)
was initiated. A regimen of 3 treatments (8 h each) with 48 hours
intervals between them was used.
[0119] Significant reduction in the prevalence and intensity of
saprolegniosis was observed after the first treatment; no further
deaths were recorded and the fish returned to their normal
behavior. The fish were visually and microscopically examined 2
days after the end of the treatments, and were found to be free of
both Saprolegnia sp. and the parasites Gyrodactilus sp. and
Trichodina sp.
Example 7
Scanning Electron Microscopy Analysis of the Effect of
Blankophor.RTM. BA on S. parasitica
Materials and Methods
[0120] S. parasitica T-1 hyphae and cysts from a 5 day RPMI-1640
broth culture, were exposed to different concentrations of
Blankophor.RTM. BA (10, 25, 100 or 1,000 mg/L) for different
periods of time. The specimens were fixed overnight in 2%
glutaraldehyde, then with 1% OSO.sub.4 for 2 h at room temperature,
and dehydrated in ascending concentrations of alcohol (25-100%) and
dried. The samples were viewed with a Leo 982 optical scanning
electron microscope at an acceleration of 6 kV.
Results
[0121] Scanning Electron Microscopy (SEM) of S. parasitica hyphae
and cysts shows that treatment with Blankophor.RTM. BA (1,000 mg/L)
results in highly distorted, wrinkled, and collapsed cell walls
(FIG. 5A), whereas the untreated control displays undamaged cell
walls (FIG. 5B). These results strongly support the conclusion that
Blankophor.RTM. BA disrupts the integrity of the cell wall in
Saprolegnia. Accordingly, the mechanism of action of
Blankophor.RTM. BA may involve one or more specific targets in the
oomycete cell wall.
[0122] Examples 8-10 describe a battery of biological toxicity
studies, carried out to verify that compounds of the invention are
non-toxic to non-target organisms.
Example 8
Toxicity of Blankophor.RTM. BA to Mice
Materials and Methods
[0123] Blankophor.RTM. BA was prepared at various concentrations in
5% dextrose and filter sterilized by passage through a sterile 0.2
.mu.m pore size cellulose acetate filter (Schleicher & Schuell,
Dassel, Germany). Male albino ICR mice (weight 30 g) were injected
through the tail vein with various doses of Blankophor.RTM. BA.
Each dosage form was administered intravenously as single bolus
injection of 0.1 ml of the same dose every 10 min to a group of 10
mice until death was observed. The survival of mice that received
the maximal tolerated dose (MTD) was monitored for 8 days.
Results
[0124] The maximal tolerated dose (MTD) of Blankophor.RTM. BA in
ICR mice was found to be >1 g/kg, confirming that it is a safe
compound, as previously determined (Stilbene Fluorescent Whitening
Agents Category, submitted to the US Environmental Protection
Agency by the ETAD Fluorescent Whitening Agent Task Force, Oct. 6,
2005).
Example 9
Toxicity of Blankophor.RTM. BA to Zooplankton
[0125] Daphnia sp. is a small (0.2 to 5 mm in length) planktonic
crustacean, commonly used as a model for aquatic toxicity.
Materials and Methods
[0126] Daphnia (.about.1,000 cells/L water) was exposed to
Blankophor.RTM. BA 100 and 1,000 mg/L). The experiment was carried
out in covered flasks (100 ml/per flask), equipped with air supply.
The flasks were incubated at room temperature for 48 h, and the
Daphnia motility was recorded.
Results
[0127] After 48 h of incubation, no difference was noted between
Daphnia that was exposed to Blankophor.RTM. BA, and the non-exposed
control group, indicating that Blankophor.RTM. BA is not toxic to
this organism.
Example 10
Toxicity of Blankophor.RTM. BA to Phytoplankton
[0128] The Delayed Fluorescent Excitation Spectroscopy (DFES)
method was used for qualitative and quantitative assessment of the
influence of Blankophor.RTM. BA on the natural population of
phytoplankton, and on specific algae strains. Delayed fluorescence
is a unique characteristic of photosynthetically active cells, as
it is an outcome of recombination that takes place in the
thylakoids in the dark (Yacobi Y. Z., V. Gerhardt, Y. Gonen-Zurgil,
and A. Sukenik. 1998. Delayed fluorescence excitation spectroscopy:
a rapid method for the qualitative and quantitative assessment of
natural population of phytoplankton. Wat. Res. 00:1-6).
Materials and Methods:
[0129] Samples from Lake Kinneret and pure cultures of Peridinium
sp. and Microcystis sp. were exposed to Blankophor.RTM. BA (100
mg/L), for a few minutes and the concentration of chlorophyll-a was
determined (in duplicate) according to the excitation spectrum
examined (range from 400 to 730 nm). In addition, the exposed and
non-exposed samples were assessed for carbon uptake with a .sup.14C
technique.
Results
[0130] Photosynthetic activity in Lake Kinneret water samples was
not deleteriously affected following exposure to Blankophor.RTM. BA
(100 mg/L), since the total chlorophyll concentration was 9.2
.mu.g/L (average), as compared to 4.6 .mu.g/L (average) prior to
exposure. Similarly, when a pure culture of Microcystis was exposed
to Blankophor.RTM. BA (100 mg/L), no difference in photosynthetic
activity was noted as compared to non-treated samples
[0131] The results of the .sup.14C-carbon uptake study, shown in
FIG. 6, are consistent with the DFES experiments, and indicate that
exposure to Blankophor.RTM. BA did not decrease the photosynthetic
activity in pure cultures of various algal species (Chlorella,
Microcystis, Peridinium and Melosira), nor in the total algae
population in Lake Kinneret. Taken together, these preliminary
findings support the conclusion that Blankophor.RTM. BA is not
toxic to phytoplankton.
CONCLUSIONS
[0132] In order to identify an effective treatment for
saprolegniosis to replace hazardous prior art treatments, such as
Malachite Green, a number of antimicrobial and antifungal agents,
detergents and disinfectants were evaluated (Table 4). Based on the
combination of results obtained in the various assay systems, and
parameters of animal safety, environmental impact and cost, each of
the compounds was assigned a utility index (UI). Of the compounds
fully evaluated, Blankophor.RTM. BA has the highest UI. This
strongly suggests that
4,4'-bis-(1,3,5-triazinylamino)stilbene-2,2'-disulfonic acid
derivatives, as provided by Blankophor.RTM. BA, can serve as
therapeutically effective, safe and economical alternatives to
Malachite Green.
TABLE-US-00004 TABLE 4 Utility of various compounds against
Saprolegnia Activity in Therapeutic Utility Compound vitro (MIC)
Toxicity.sup.1 efficacy Cost Index.sup.2 (UI) Hydrogen High
Moderate ND Low Moderate peroxide (10 mg/L) (problematic in earth
pond) Sodium Extremely ND ND High Low chloride Low (30,000 mg/L)
Sodium High High (in ND Low Low percarbonate (10 mg/L) low
temperature) Detergents, Variable High Non effective Low Low
including Agrosept Formaldehyde Moderate Moderate Effective Low Low
(negative (100 mg/L) safety and environmental profile) Pyceze .RTM.
Moderate Moderate Non effective High Low (100 mg/L) Amphotericin
High Moderate ND High Low B (0.1-0.5 mg/L) Blankophor .RTM.
Moderate Low Very Effective Low High BA (100 mg/L) .sup.1Toxicity
was defined as: high = 50 ppm; moderate = 50-200 ppm; and low =
>1000 ppm. .sup.2Utility Index (UI): Index measuring the
usefulness of the compound by considering the parameters of
toxicity, therapeutic efficacy and cost.
[0133] While certain embodiments of the invention have been
illustrated and described, it will be clear that the invention is
not limited to the embodiments described herein. Numerous
modifications, changes, variations, substitutions and equivalents
will be apparent to those skilled in the art without departing from
the spirit and scope of the present invention as described by the
claims, which follow.
* * * * *